ABSTRACT Vitamin K epoxide reductase (VKOR) is a membrane-embedded enzyme and the target of warfarin, the most commonly used oral anticoagulant. Due to limited structural knowledge, there is a fundamental gap in understanding the mechanism of VKOR catalysis and warfarin inhibition. Filling this knowledge gap will provide the basis to produce clotting factors for therapeutic use and to design novel drugs regulating blood clotting. Our long-term goal is to understand the entire vitamin K cycle that supports blood coagulation. The cycle begins with the carboxylation of designated glutamate residues in coagulation factors, a modification required for calcium-dependent activation at sites of injury. The carboxylation reaction consumes vitamin K hydroquinone, which is regenerated by VKOR's reduction of vitamin K epoxide. The reducing equivalents may come from VKOR's partners that transfer electrons to VKOR to maintain its activity. The objective of this application is to use structural biology, biochemistry, and cell biology approaches to elucidate the mechanisms of VKOR catalysis, partner interaction, and warfarin inhibition. Our hypotheses are 1) VKOR uses distinctive catalytic mechanisms for vitamin K reduction and electron transfer, 2) the VKOR-partner interaction is important to support blood coagulation, and 3) unique features at the active site of eukaryotic VKORs confer the epoxide-reductase activity and the warfarin sensitivity. These hypotheses are based on preliminary data produced in the applicant's laboratory. 1) We have determined a new 2.8 structure of a VKOR homolog. The structure reveals a hydrophobic pocket at the active site that may increase the reactivity of a catalytic cysteine, and electron transfer is facilitated by the unwinding motion of a designated helix. 2) Using a cell-based assay, we have identified partner proteins that can stimulate the production of carboxylated coagulation factor. 3) We have determined a low-resolution structure of a VKOR homolog in complex with a warfarin analog. In addition, our biochemical experiments have provided a new explanation for warfarin resistance. The hypotheses will be tested by three specific aims. 1) We will determine crystal structures of the VKOR homolog captured in different conformational states during the electron transfer. The structural knowledge will guide our functional studies in human VKOR to elucidate the mechanisms controlling its catalytic efficiency and electron flux. 2) We will assess the ability of partner proteins to support the in vivo and in vitro activity of human VKOR. 3) We will determine the structures of eukaryotic VKORs, and the structures of VKORs in complex with warfarin analogs. We will also elucidate the mechanism of warfarin inhibition and resistance by biochemical studies and by other physical methods to probe the drug binding.